1. Field of the Invention
The present invention relates generally to the fields of protein biochemistry and fluorescence resonance energy transfer. More specifically, the invention relates to the creation and use of novel cyan fluorescent protein variants with improved fluorescent characteristics.
2. Description of Related Art
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems (Tsien, 1998). Over the last several years, both random and semi-rational mutagenesis have produced GFP variants with new colors, improved folding properties, increased brightness, and altered pH-sensitivity. Through genetic manipulations, hundreds of proteins have been successfully fused to GFPs to allow monitoring of their expression and trafficking. When GFP or GFP-fusion proteins are heterologously expressed at a certain level, the intensity of the fluorescence depends on: (1) the ultimate brightness of GFP fluorophore, which is limited by the product of extinction coefficient and fluorescence quantum yield; (2) the maturation efficiency of newly-synthesized GFP polypeptides; and (3) the extent of quenching of GFP fluorophore by environmental factors.
Yellow fluorescent protein (YFP) is one of the most commonly used GFP variants and has the longest wavelength emission of all Aequorea GFP variants, and EYFP, containing the modification S65G, S72A and T203Y, is a common variant of YFP. The extinction coefficient and fluorescence quantum yield of most YFP variants are within 60,000 to 100,000 M−1 cm−1 and 0.6 to 0.8, respectively (Tsien, 1998). These values are almost comparable to those of common bright fluorophores, such as fluorescein and rhodamine. Therefore, the improvement of the ultimate brightness of YFP seems to have reached its limit.
Newly-synthesized GFP polypeptides need to mature properly before emitting fluorescence. The maturation involves two steps: first, the protein folding into a nearly native conformation, and then, cyclization of an internal tripeptide followed by oxidation. Some of the primary mutations that improve maturation of GFP have been identified (Tsien, 1998). For example, F64L, M153T, V163A, and S175G are common mutations introduced in many enhanced GFP variants. M153T and S175G are located on the surface of the β-barrel and are known to enhance the folding efficiency and the stability by reducing surface hydrophobicity and increasing the solubility of the protein.
Another study generated, using random mutagenesis on pericams (circularly-permuted GFPs engineered to sense Ca2+), generated several mutations that improved the maturation without affecting the Ca2+-sensitivity. Of particular interest was a mutation of Phe-46 to Leu, which greatly improved the formation of the chromophore at 37° C. The effect of the well-known folding mutation, F64L/M153T/V163A/T203Y, on EYFP was also studied along with that of F46L. The purified YFP variants exhibited exactly the same excitation and emission spectra. However, the F46L mutant also gave rise to about 20-fold increase in the fluorescence of cell pellet after 12 hr incubation. Both SEYFP and SEYFP-F46L refolded quickly with rate constants. Although F46L alone increased the speed and yield of recovery of EYFP and SEYFP, its effect was less potent than that of the common folding mutations. Thus, it was concluded that the mutations F64L/M153T/V163A/S175G were significantly effective in facilitating folding of YFP at 37° C. While SEYFP and SEYFP-F46L gave similar folding rate constants, the speed and yield of the renaturation from denatured/reduced protein at 37° C. was significantly improved by F46L. Interestingly, this improvement was not clearly observed when the studies were carried out at room temperature. Also, EYFP-F46L showed faster reoxidation than SEYFP at 37° C. This discussion illustrates that fluorescent variants can be engineered to, exhibit one or more beneficial properties selected from improved maturation speed, accelerated oxidation step, and decreased pH-sensitivity, each of which can lead to the enhancement of fluorescence development.
Thus, fluorescent proteins clearly are amenable to considerable engineering, and can be manipulated such that the variants exhibit additional beneficial properties not found in the natural molecules or existing variants. Therefore, there is an opportunity and need to create new and improved fluorescent proteins for a variety of uses.